PROJECT SUMMARY/ABSTRACT T cells demonstrate remarkable sensitivity to activating TCR signals, yet preserve controlled homeostasis in the basal state. How T cells balance these contrasting features is not known. T cells express two types of Ras Guanine nucleotide Exchange Factors (RasGEFs), SOS1 and RasGRP1, and we have established that these RasGEFs are autoinhibited and activated in unique manners. Surprisingly, the roles of SOS1 and RasGRP1 in peripheral T cells have not been studied. Based on findings made during our ongoing P01 program, we hypothesize that these RasGEFs are non-redundant to allow for concurrent basal and TCR-induced RasGEF signals to control T cell homeostasis while enabling proper and efficient T cell activation and polarization. This proposal is a renewal of an ongoing P-01 program. Project 2 will focus on understanding the roles of SOS1 and RasGRP1 in canonical Ras signaling and non-canonical RasGEF signaling in peripheral T cells, downstream of the kinase module studied in Project 1, and how these signals establish the balance between homeostasis and sensitive receptor-triggered T cell activation. We will combine biophysical, computational, biochemical, and immunology expertise of the Kuriyan (UCB), Groves (UCB), Chakraborty (MIT), and Roose (UCSF) labs. As in our ongoing P01 program, a constant iteration between the synergistic approaches represented in four different groups will lead to broader insights than from individual efforts. Our expertise spans structural biochemistry, mass spectrometry, and computational models, to quantitative biochemical measurements of Ras activation on model membrane systems, live cell RasGEF-Ras imaging, mouse models, and human disease RasGEF variants. The overall goals of project #2 are (a) to understand how basal and TCR-induced RasGEF signaling regulates the primed but controlled state while allowing for efficient peripheral T cell activation and (b) how non-synonymous variants (SNPs) in human SOS1 and RasGRP1 impact autoinhibition, activation, and termination of RasGEF signaling, and thereby perturb balanced T cell homeostasis and function. Capitalizing on new insights and novel tools described in the proposal, we will test our hypothesis that RasGRP1 dominates the early and basal phase of Ras signals in T cells followed by a high-gain SOS- dominated phase with kinetic proofreading functions. In Aim 1, we will determine the molecular mechanisms of RasGEF autoinhibition. In Aim 2, we will resolve the biochemical and spatial aspects of TCR-induced RasGEF activation and termination. In Aim 3, we will determine the roles of canonical Rasgrp1 and SOS1 Ras-ERK MAPK signaling for peripheral T cell function. In Aim 4, we will define the role of non-canonical RasGEF signals in peripheral T cells. Through our collaborative studies we anticipate to uncover how full length SOS1 and RasGRP1 are autoinhibited, how enzymatic- and adapter- functions of these Ras exchange factors are regulated and impact basal and TCR-induced canonical Ras-ERK and non-canonical P38 and mTOR pathways and T cell function, and how SOS1 and RasGRP1 variants with altered regulation cause T cell pathology.